Chap 5-Techniques in protein Biochemistry

Question 1

The proteome def

Answer 1

The level of functional information, which encompasses the types, functions, and interactions of proteins that yield a functional unit. name comes from, proteins expressed by the genome.

Question 2

What does the genome tell us?

Answer 2

The genome provides a list of gene products that could be present but only a subset will actually be expressed in a given biological context. It is a fixed characteristic of the cell.

Question 3

WHat does the proteome tell us?

Answer 3

The proteome tells us what proteins aee functionally present. it is not a fixed characteristic of the cell. Varies with cell type, developmental stage, and environmental conditions such as presence of hormones,

Question 4

What do we have to do before we attempt to purify a protein?

Answer 4

We must perform a test that identifies the protein we are interested in. The test is called an assay, and it is based on some unique identifying properties of the protein. the test is used after each stage of purification to see if it’s working.

Question 5

How can we be sure that our purification scheme is working?

Answer 5

We need to know the total protein present in the mixture being assayed, This measurement includes the enzyme of interest and all other proteins present, but doesnt tell us about enzyme activity. We can assess the progress of purification by measuring the specific activity: the ratio of enzyme activity to the amount of protein in the enzyme assay.

Question 6

WHat is the point of the purification

Answer 6

to remove allproteins except the protein in which we are interested. Quantitatively, we want to maximize specific activity.

Question 7

What is a homogenate?

Answer 7

It is a mixture of all of the components of the cell but no intact cells. caused by a disruption of the cell membranes.

Question 8

What is the supernatant

Answer 8

the lighter solution above when the mixture is centrifuged,

Question 9

What are the pellets

Answer 9

heavy material found at the bottom of the centrifuge tube. The pellects are enriched in a particular organelle.

Question 10

differential centrifugation def

Answer 10

The supernatant from the first centrifugation is centrifuged again at greater force to yield another pellet and supernatant, this yields several fractions of decreasing density, each still containing hundreds of different proteins,

Question 11

Salting out def

Answer 11

Most proteins precipitate out of solution at high salt concentrations. Salting out is due to a competition between the salt ions and the protein for water to keep the protein in solution. The salt concentration differs for different proteins. Many proteins lose their activity in such high salt concentrations.

Question 12

dialysis def

Answer 12

The process of removing salt. The protein-salt solution is placed in a small bag made of semipermeable membrane that proteins are too big to move out but ions and salts can.

Question 13

Molecular exclusion Chromatography

Answer 13

also called gel-filtration chromatography, separates proteins based on size. Small molecules can enter the porous beads making up the column, whereas large ones cannot.

Question 14

Ion exchange Chromatography

Answer 14

Proteins are separated based on their net charge. If a protein has a positive charge at pH 7, it will usually bind to a column of beads containing negatively charged carboxylate groups. The positively charged proteins can then be released by increasing the concentration of salt in the buffer poured over the column.

Question 15

Affinity chromatography

Answer 15

Some proteins have a high affinity for specific chemical groups or specific molecules, For example, protein concanavalin A binds to glucose reversibly, can be purified by passing an extract through a column of beads containing glucose residues.

Question 16

High pressure liquid chromatography

Answer 16

Enhanced version of the other column techniques. The beads in the column are more finely divided and so there are more interaction sites and thus greater resolving power. Pressure must be applied to obtain adequate flow rates.

Question 17

gel electrophoresis

Answer 17

Allows us to visualize the number of proteins present at each step. A molecule with a net charge will move in an electric field- electrophoresis. Molecules that are small compared with the pores in the gel readily move through the gel , whereas larger molecules are almost immoblie. The pH of the electrophoretic olution is adjusted so that all of the proteins are negativeky charged

Question 18

What is the direction of flow of molecules in gel electrophoresis?

Answer 18

From the cathode, negative charge to the anode, positive charge.

Question 19

How can proteins be separated by mass?

Answer 19

by electrophoresis is a polyacrylamide gel in the presence of the detergent sodium dodecyl sulfate (SDS). Technique is called SDS-PAGE. Small proteins move rapidly through the gel, whereas large proteins stay at the top.

Question 20

What is the isoelectric point of a protein?

Answer 20

It is the pH at which its net charge is zero. At this pH, the protein will not migrate om an electric field.

Question 21

Isoelectric focusing

Answer 21

proteins move until they reach a position in the gel at which the pH is equal to their isoelectric point, the net charge of the protein is zero.

Question 22

Two dimensional electrophoresis

Answer 22

Proteins are separated in the horizontal direction based on isoelectric point and in the vertical direction based on mass. Proteins with the same pl value are now separated by mass

Question 23

Gradient centrifugation

Answer 23

Provides a convenient means of detection.

Question 24

The rate at which these complexes move when exposed to such a force, centrifugal force, is determined by?

Answer 24

Mass, shape and density. A way to calculate the rate of movement is to calculate the sedimentation coefficient. The smaller the coefficient, the more slowly a molecule moves in a centrifugal field.

Question 25

antibody def

Answer 25

a protein , synthesized by an animal in response to the presence of a foreign substance, called an antigen. Antibodies have specific and high affinity for the antigens that elicited their synthesis.

Question 26

Antigenic determinant or epitope def

Answer 26

Specific group or cluster of amino acids on a protein antigen that the antibody recognizes.

Question 27

why can it be helpful that antibodies are heterogeneous?

Answer 27

The heterogeneity of antibodies can be advantageous for the detection of a protein of low abundance because each protein molecule can be bound by more than one antibody at multiple distinct antigenic sites.

Question 28

immunoprecipitation

Answer 28

a technique where the material bound to the antibody has high affinity for the proteins moving over the matrix.

Question 29

Enzyme-linked immunosorbent assay (ELISA)

Answer 29

This method makes use of an enzyme that reacts with a colourless substrate to produce a coloured product. ELISA is rapid and can detect less than a nanogram of a protein. It works with both polyclonal or monoclonal antibodies but monoclonal has more reliable results. Purified viral core proteins are adsorbed on the well. Antiodies from the person being tested are then added to the well. Someone who is infected will have antobodies. Finally, enzyme-linked antobodies to human antibodies are allowed to react in the well, and unbound antibodies are removed by washing. Substrate is then applied. An enzyme reaction tells us that the enzyme-linked antibodies were bound to human antibodies, and the person has HIV.

Question 30

Indirect ELISA

Answer 30

Used to detect the presence of antibody and is the basis of the test for HIV.

Question 31

The sandwich ELISA

Answer 31

Used to detect antigen rather than antibody. Process: The antibody to a particular antigen is adsorbed to a well, called the captured antibody, next blood or urine contaning the antigen is added and binds to the antibody. Then, a different antibody to the antigen, the detection antibody, is added. This antibody is enzyme0linked and is processed as described for ELISA. The extent of colour formation is directly proportional to the amount of antigen present, Permits measurements small quantities of antigen. EX, home pregnancy tests

Question 32

Western blotting

Answer 32

another immunoassay technique. Allows the detection of very small quantities of a particular protein in a cell or in body fluid, It alsoo allows the determination of size of the target protein.

Question 33

Process of western blottong

Answer 33

Proteins on an SDS-polyacrylamide gel are transferred to a polymer sheet and stained with fluorecent-labeled antibody, The antibody is excited by light and the band corresponding to the protein to which the antibody bonds is visualized with an appropriate detector.

Question 34

STeps to sequencing a simple peptide.

Answer 34

1. determine the amino acid composition 2.The peptide is hydrolyzed into its amino acids by heating it in strong acid. 3.The individual amino acids can be separated by ion-exchange and visualized by treatment with fluorescamine, which reacts with the alpha-amino group to form a highly fluorescent product. 4. The solution is then run through a column. The amount of buffer required to remove the amino aicd from the column is compared with the elution pattern of a standard, revealing the identity of the amino acids in the solution.

Question 35

Edman degradation

Answer 35

Determines the sequence of a protein by sequentially removing one residue at a time from the amino end of a peptide. Have to break the protein into smaller peptides that can be sequenced independently.

Question 36

Mass spectrometry def

Answer 36

Analyzes ionized forms of molecules in the gas phase or volatile liquids. Mass measurements are obtained by determining how readily an ion is accelerated in an applied electric field.

Question 37

matrix-assisted laser desorption-ionization (MALDI)

Answer 37

In MALDI, the protein or peptide under study is coprecipitated with an organic compound that absorbs laser light of an appropriate wavelength. After gas-phase ions have been generated, several approaches may be used to determine the mass.

Question 38

Time of flight (TOF)

Answer 38

Determining mass of a protein. The ions are accelerated in an electric field toward a detector. The lighter ions are accerated more, travel faster, and arrive at the detector first. MALDI-TOF is an accurate means of determining protein mass.

Question 39

Why is an assay needed to purify a protein?

Answer 39

An assay identifies the desired protein. The ability to identify the protein is important in determining if particular purification steps are effective in isolating the protein from the other cellular material.

Question 40

Why do proteins precipitate at high salt concentrations?

Answer 40

When the salt concentration becomes too high, the salt ions interact with water molecules and then there arent enough water molecules to interact with the protein, and the protein precipitates.

Question 41

Why do some proteins require salt to dissolve?

Answer 41

When a protein solution lacks salt, the proteins may interact with one another- positive charges in one proteins and negative on another. This aggregate becomes too large to be soluble in water alone and requires the addition of salt to neutralize the charges on the proteins, preventing them from interacting with one another.

Question 42

The detergent sodium dodecyl sulfate denatures proteins, How?

Answer 42

The long hydrophobic tail on the SDS molecule disrupts the hydrophobic interaction in the interior of the protein. The protein unfolds, with the hydrophobic R groups now interacting with SDS instead of one another.

Question 43

Explain how proteins treated with a sulfhydryl reagent and dissolved in SDS have the same charge to mass ratio.

Answer 43

Because one SDS molecule bonds to a protein for every two amino acids in the proteins, in principle, all amino acids will have the same charge to mass ratio. This statement might be incorrect if the protein contains many charged amino acids.

Question 45

WHat unique property of the estrogen receptor allows for its identification and purification?

Answer 45

The estrogen receptor has a unique, high affinity for the estrogen estradiol

Question 46

Antigen

Answer 46

foreign biomolecules

Question 47

antigenic determinant

Answer 47

- molecular feature recognized by antibodies -epitopes

Question 48

polyclonal antibodies

Answer 48

-Immunoglobins -heterogeneous antibodies -antibodies produced by the injection of a biomolecule into an animal

Question 49

monoclonal antibodies

Answer 49

- immunoglobins -antibodies produced by hybridoma cells - homogeneous antibodies

Question 50

Differenciate between polyclonal and monoclonal antibodies

Answer 50

Polyclonal antibodies are a collection of heterogeneous antibodies that bind to multiple epitopes on an antigen Monoclonal antibodies are homogeneous antibodies and they constitute a collection of antibodies that bind to a single epitope on an antigen

Question 51

Explain how immunoprecipitation can be used to purify proteins

Answer 51

If there exists an antibody to a protein, the antibody can be attached to an insoluble bead of some sort. A mixture of proteins that includes the wanted protein is mixed with the antibody, Only the protein of interest binds to the highly specific antibody. The mixture is centrifuged, and the supernatant, top layer, is discarded. The protein of interest is then released from the antibody by adding a protein denaturant.

Question 52

What is an ELISA and how is it used?

Answer 52

An enzyme-linked immunoabsorbant assay ELISA is used for quantitating the presence of an antigen by using an enzyme linked to an antibody to the antigen. How much antigen/protein there is.

Question 53

Describe western blotting

Answer 53

Western blotting is used to detect a specific protein in a cell or body fluid by subjecting a sample to SDS-polyacrylamide electrophoresis to resolve the proteins, which are then transferred, or blotted, to a polymer sheet where an antibody specific for the protein we want is incubated with the sample. Other enzyme-linked antibodies can then be used to visualize the desired antibody-antigen complex.

Question 54

Why can the Edman method only sequence proteins that are 50 amino acids long?

Answer 54

After the 50 repetitions, many different peptides release different amino acids at the same time and the efficiency of this technique drastically drops.

Question 55

Mass spectrometry often does not allow its unique identification among all possible proteins but by using the masses of all fragments produced by digestion with trypsin allows unique identification. Why?

Answer 55

Many proteins have similar masses but different sequences and different patterns when digested with trypsin. The set of masses of tryptic peptides forms a detailed fingerprint of a protein that is very unlikely to appear at random in other proteins, regardless of size.